u-0126 and Hypoxia

Obstructive sleep apnea syndrome (OSAS) is a common and complex disorder that is associated with liver injury. Moreover, previous studies have revealed that chronic intermittent hypoxia (CIH) is associated with the development of non‑alcoholic fatty liver disease and hepatic fibrosis. However, the underlying molecular mechanisms remain largely unknown. The present study aimed to investigate whether chronic intermittent hypoxia induced hepatic fibrosis, in addition to determining its underlying mechanisms, in CIH model rats using immunohistochemistry, western blotting and reverse transcription‑quantitative PCR. The present results suggested that CIH caused hepatic fibrosis and increased the expression levels of interleukin (IL)‑1β, IL‑8, monocyte chemotactic‑1, tumor necrosis factor‑α, intercellular adhesion molecule‑1 and vascular cell adhesion molecule‑1 in the liver; these conditions could be reversed by Toll‑like receptor 4 (TLR4) short hairpin RNA lentivirus treatment. Moreover, immunohistochemistry and western blotting results indicated that TLR4 and NF‑κB expression levels were significantly increased in the CIH and CIH‑TLR4 empty vector lentivirus group. However, protein expression levels of TLR4, NF‑κB, inhibitor of NF‑κB and phosphorylated‑mitogen‑activated protein kinase (MAPK)‑1 in the hypoxia/reoxygenation group were significantly higher compared with the control group (P<0.05), and these results were reversed by the MAPK inhibitor U0126 in vitro. Collectively, the present preliminary results suggested that inflammation and the TLR4/NF‑κB/MAPK signaling pathway may be involved in CIH‑induced liver fibrosis.

Topics: Animals; Butadienes; Cell Line; Disease Models, Animal; Enzyme Inhibitors; Gene Silencing; Hepatic Stellate Cells; Hypoxia; Inflammation; Liver Cirrhosis; Male; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Rats; Rats, Sprague-Dawley; Signal Transduction; Sleep Apnea, Obstructive; Toll-Like Receptor 4

We previously demonstrated decreased expression of key genes regulating cortisol biosynthesis in long-term hypoxic (LTH) sheep fetal adrenals compared to controls. We also showed that inhibition of the extracellular signal-regulated kinases (ERKs) with the mitogen-activated protein kinase (MEK)/ERK inhibitor UO126 limited adrenocorticotropic (ACTH)-induced cortisol production in ovine fetal adrenocortical cells (FACs), suggesting a role for ERKs in cortisol synthesis. This study was designed to determine whether the previously observed decrease in LTH cytochrome P45011A1/cytochrome P450c17 (CYP11A1/CYP17) in adrenal glands was maintained in vitro, and whether ACTH alone with or without UO126 treatment had altered the expression of CYP11A1, CYP17, and steroidogenic acute regulatory protein (StAR) in control versus LTH FACs. Ewes were maintained at high altitude (3820 m) from ∼40 days of gestation (dG). At 138 to 141 dG, fetal adrenal glands were collected from LTH (n = 5) and age-matched normoxic controls (n = 6). Fetal adrenocortical cells were challenged with ACTH (10

Topics: Adrenal Cortex; Adrenocorticotropic Hormone; Animals; Butadienes; Cell Survival; Enzyme Inhibitors; Hydrocortisone; Hypoxia; Nitriles; Phosphoproteins; Sheep

Topics: Animals; Animals, Newborn; Butadienes; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Protein-delta; Cobalt; Diabetic Retinopathy; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; MAP Kinase Signaling System; Neovascularization, Pathologic; Nitriles; Proto-Oncogene Proteins c-fos; Rats; Receptors, G-Protein-Coupled; Retina; Vascular Endothelial Growth Factor A

The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here, we provide evidence that upon activation by an agonist, H3Rs trigger ERK1/2 activation via phospholipase C/protein kinase C (PLC/PKC)-, phospholipase D (PLD)s-, and matrix metallopeptidase/epidermal growth factor receptor (MMP/EGFR) transactivation-dependent pathways. Moreover, we demonstrate that H3Rs exhibit a neuroprotective effect on the cultured mouse cortical neurons under hypoxia conditions through the ERK1/2 pathway.

Topics: Animals; Animals, Newborn; Butadienes; Cells, Cultured; Cerebral Cortex; Chlorocebus aethiops; Enzyme Inhibitors; ErbB Receptors; Glucose; Histamine; Humans; Hypoxia; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Sequence Data; Neurons; Nitriles; Pertussis Toxin; Phospholipase D; Protein Kinase C; Receptors, Histamine H3; Signal Transduction; Time Factors

Erythropoietin stimulation modulates the central respiratory command in newborn mice. Specifically, the central respiratory depression induced by hypoxia is attenuated by acute (1h) or abolished by chronic erythropoietin stimulation. However, the underlying mechanisms remain unknown. As MEK and PI3K pathways are commonly involved in Epo-mediated effects of neuroprotection and erythropoiesis, we investigated here the implication of PI3K and MEK1/2 in the Epo-mediated regulation of the central respiratory command. To this end, in vitro brainstem-spinal cord preparations from 3 days old transgenic (Tg21; constitutively overexpressing erythropoietin in the brain specifically) and control mice were used. Our results show that blockade of PI3K or MEK1/2 stimulates normoxic bursts frequency in Tg21 preparations and abolish hypoxia-induced frequency depression in control preparations. These results show that MEK1/2 and PI3K pathways are involved in the Epo-mediated regulation of the central respiratory command. Moreover, this is the first demonstration that MEK1/2 and PI3K are involved in the brainstem central respiratory command.

Topics: Animals; Animals, Newborn; Butadienes; Central Nervous System; Chromones; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythropoietin; Hypoxia; In Vitro Techniques; Long-Term Synaptic Depression; MAP Kinase Kinase 1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Motor Neurons; Nitriles; Phosphatidylinositol 3-Kinases; Respiration; Signal Transduction

The carotid body is the main mammalian oxygen-sensing organ regulating ventilation. Despite the carotid body is subjected of extensive anatomical and functional studies, little is yet known about the molecular pathways signaling the neurotransmission and neuromodulation of the chemoreflex activity. As kinases are molecules widely involved in motioning a broad number of neural processes, here we hypothesized that pathways of protein kinase B (AKT) and extracellular signal-regulated kinases ½ (ERK1/2) are implicated in the carotid body response to hypoxia. This hypothesis was tested using the in-vitro carotid body/carotid sinus nerve preparation ("en bloc") from Sprague Dawley adult rats. Preparations were incubated for 60 min in tyrode perfusion solution (control) or containing 1 μM of LY294002 (AKT inhibitor), or 1 μM of UO-126 (ERK1/2 inhibitor). The carotid sinus nerve chemoreceptor discharge rate was recorded under baseline (perfusion solution bubbled with 5 % CO(2) balanced in O(2)) and hypoxic (perfusion solution bubbled with 5 % CO(2) balanced in N(2)) conditions. Compared to control, both inhibitors significantly decreased the normoxic and hypoxic carotid body chemoreceptor activity. LY294002- reduced carotid sinus nerve discharge rate in hypoxia by about 20 %, while UO-126 reduces the hypoxic response by 45 %. We concluded that both AKT and ERK1/2 pathways are crucial for the carotid body intracellular signaling process in response to hypoxia.

Topics: Animals; Butadienes; Carotid Body; Chromones; Hypoxia; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Nitriles; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley

Bone marrow-derived mesenchymal stem cells (bmMSCs) are the most promising seed cells for cell transplant therapy. Hypoxia is a known stimulus of autophagy. Recent studies showed that hypoxia promotes autophagy of human placental chorionic plate-derived mesenchymal stem cells (CP-MSCs). However, whether hypoxia affects autophagy of bmMSCs has not been examined. The goal of this study was to investigate the effect of hypoxia on autophagy of mouse bmMSCs and the underlying mechanisms.. BmMSCs from mouse bone marrow were randomly divided into three groups: control (C), hypoxia (H) and hypoxia + reoxygenation (H+R) groups. Subsequent autophagic signals were analyzed by immunostaining and Western blot assays.. The expression of autophagic signals LC-3, Atg5 and Beclin-1, as well as the conversion of LC3B-I to LC3B-II in bmMSCs were significantly increased in H group as compared with control (p<0.05). These autophagic signals were also higher in H+R group than in H group (p<0.05). Also, the expression of phospho-ERK1/2 was significantly increased in H and H+R groups as compared with control (p<0.05). Notably, application of ERK1/2 inhibitor U0126 (5μM) significantly repressed hypoxia-induced expression of LC-3 and Atg5, as well as conversion of LC3B-I to LC3B-II (p<0.05).. Hypoxia can induce autophagy of bmMSCs, which depends on activation of ERK1/2 pathway.

Topics: Animals; Autophagy; Bone Marrow Cells; Butadienes; Cells, Cultured; Enzyme Activation; Hypoxia; Mesenchymal Stem Cells; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles

Our previous data indicated that hypoxic preconditioning (HPC) ameliorates transient global cerebral ischemia (tGCI)-induced neuronal death in hippocampal CA1 subregion of adult rats. However, the possible molecular mechanisms for neuroprotection of this kind are largely unknown. This study was performed to investigate the role of the mitogen-activated protein kinase/extra-cellular signal-regulated kinase kinase (MEK)/extra-cellular signal-regulated kinase (ERK) pathway in HPC-induced neuroprotection. tGCI was induced by applying the four-vessel occlusion method. Pretreatment with 30 min of hypoxia applied 1 day before 10 min tGCI significantly decreased the level of MEK1/2 and ERK1/2 phosphorylation in ischemic hippocampal CA1 subregion. Also, HPC decreased the expression of phosphorylated ERK1/2 in degenerating neurons and astrocytes. However, the administration of U0126, a MEK kinase inhibitor, partly blocked MEK1/2 and ERK1/2 phosphorylation induced by tGCI. Meanwhile, neuronal survival was improved, and glial cell activation was significantly reduced. Collectively, these data indicated that the MEK/ERK signaling pathway might be involved in HPC-induced neuroprotection following tGCI. Also, HPC resulted in a reduction of glial activation.

Topics: Aging; Animals; Brain Ischemia; Butadienes; CA1 Region, Hippocampal; Cell Death; Cytoprotection; Hypoxia; Male; MAP Kinase Signaling System; Neurons; Neuroprotective Agents; Nitriles; Phosphorylation; Rats; Rats, Wistar

Myocardial preconditioning is a powerful phenomenon that can attenuate ischemia/reperfusion-induced oxidant stress and elicit delayed cardioprotection. Its mechanisms involve activation of intracellular signaling pathways and up-regulation of the protective antioxidant proteins. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in attenuating oxidant stress and promoting cell survival. In the present study, we investigated whether DJ-1 is up-regulated during the late phase of hypoxic preconditioning (HP) and the up-regulation of DJ-1 is mediated by extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway. Rat heart-derived H9c2 cells were exposed to HP. Twenty-four hours later cells were subjected to hypoxia/reoxygenation (H/R) and then cell viability, lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), ERK1/2 phosphorylation, and DJ-1 protein were measured appropriately. The results showed that HP efficiently attenuated H/R-induced viability loss and LDH leakage. In addition, HP promoted ERK1/2 activation, up-regulated DJ-1 protein expression, inhibited H/R induced the elevation of ROS. However, when ERK1/2 phosphorylation was specifically inhibited by U0126, the increase in DJ-1 expression occurring during HP was almost completely abolished and, as a result, the delayed cardioprotection induced by HP was abolished, and the inhibitory effect of HP on H/R-induced oxidant stress was also reversed. Furthermore, knocking down DJ-1 by siRNA attenuated the delayed cardioprotection induced by HP. Our data indicate that HP can up-regulate DJ-1 protein expression through the ERK1/2-dependent signaling pathway. Importantly, DJ-1 might be involved in the delayed cardioprotective effect of HP against H/R injury.

Topics: Animals; Butadienes; Cell Line; Cell Survival; Enzyme Activation; Gene Knockdown Techniques; Hypoxia; L-Lactate Dehydrogenase; MAP Kinase Signaling System; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardium; NF-kappa B; Nitriles; Oxygen; Protein Deglycase DJ-1; Rats; Reactive Oxygen Species; RNA, Small Interfering; Time Factors; Up-Regulation

Acute intermittent hypoxia (AIH) elicits a form of spinal respiratory plasticity known as phrenic long-term facilitation (pLTF). pLTF requires spinal serotonin receptor-2 activation, the synthesis of new brain-derived neurotrophic factor (BDNF), and the activation of its high-affinity receptor tyrosine kinase, TrkB. Spinal adenosine 2A receptor activation elicits a distinct pathway to phrenic motor facilitation (pMF); this BDNF synthesis-independent pathway instead requires new synthesis of an immature TrkB isoform. Since hypoxia increases extracellular adenosine levels, we tested the hypothesis that new synthesis of TrkB and BDNF contribute to AIH-induced pLTF. Furthermore, given that signaling mechanisms "downstream" from TrkB are unknown in either mechanism, we tested the hypothesis that pLTF requires MEK/ERK and/or phosphatidylinositol 3-kinase (PI3K)/Akt activation. In anesthetized Sprague-Dawley rats, an intrathecal catheter at cervical level 4 was used to deliver drugs near the phrenic motor nucleus. Since pLTF was blocked by spinal injections of small interfering RNAs targeting BDNF mRNA but not TrkB mRNA, only new BDNF synthesis is required for AIH-induced pLTF. Pretreatment with a MEK inhibitor (U0126) blocked pLTF, whereas a PI3K inhibitor (PI-828) had no effect. Thus, AIH-induced pLTF requires MEK/ERK (not PI3K/AKT) signaling pathways. When U0126 was injected post-AIH, pLTF development was halted but not reversed, suggesting that ERK is critical for the development but not maintenance of pLTF. Thus, there are clear mechanistic distinctions between AIH-induced pLTF (i.e., BDNF synthesis and MEK/ERK dependent) versus adenosine 2A receptor-induced pMF (i.e., TrkB synthesis and PI3K/Akt dependent).

Topics: Animals; Blood Gas Analysis; Brain-Derived Neurotrophic Factor; Butadienes; Hypoxia; Injections, Spinal; Long-Term Potentiation; Male; MAP Kinase Signaling System; Neuronal Plasticity; Nitriles; Phosphatidylinositol 3-Kinases; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Receptor, trkB; Receptors, Adenosine A2; Respiratory System; RNA, Messenger; Signal Transduction; Spinal Cord

Endothelial cells (EC) serve a paracrine function to enhance signaling in cardiomyocytes (CM), and conversely, CM secrete factors that impact EC function. Understanding how EC interact with CM may be critically important in the context of ischemia-reperfusion injury, where EC might promote CM survival. We used isoflurane as a pharmacological stimulus to enhance EC protection of CM against hypoxia and reoxygenation injury. Triggering of intracellular signal transduction pathways culminating in the enhanced production of nitric oxide (NO) appears to be a central component of pharmacologically induced cardioprotection. Although the endothelium is well recognized as a regulator for vascular tone, little attention has been given to its potential importance in mediating cardioprotection. In the current investigation, EC-CM in co-culture were used to test the hypothesis that EC contribute to isoflurane-enhanced protection of CM against hypoxia and reoxygenation injury and that this protection depends on hypoxia-inducible factor (HIF1α) and NO. CM were protected against cell injury [lactate dehydrogenase (LDH) release] to a greater extent in the presence vs. absence of isoflurane-stimulated EC (1.7 ± 0.2 vs. 4.58 ± 0.8 fold change LDH release), and this protection was NO-dependent. Isoflurane enhanced release of NO in EC (1103 ± 58 vs. 702 ± 92 pmol/mg protein) and EC-CM in co-culture sustained NO release during reoxygenation. In contrast, lentiviral mediated HIF1α knockdown in EC decreased basal and isoflurane stimulated NO release in an eNOS dependent manner (517 ± 32 vs. 493 ± 38 pmol/mg protein) and prevented the sustained increase in NO during reoxygenation when co-cultured. Opening of mitochondrial permeability transition pore (mPTP), an index of mitochondrial integrity, was delayed in the presence vs. absence of EC (141 ± 2 vs. 128 ± 2.5 arbitrary mPTP opening time). Isoflurane stimulated an increase in HIF1α in EC but not in CM under normal oxygen tension (3.5 ± 0.1 vs. 0.79 ± 0.15 fold change density) and this action was blocked by pretreatment with the Mitogen-activated Protein/Extracellular Signal-regulated Kinase inhibitor U0126. Expression and nuclear translocation of HIF1α were confirmed by Western blot and immunofluorescence. Taken together, these data support the concept that EC are stimulated by isoflurane to produce important cardioprotective factors that may contribute to protection of myocardium during ischemia and reperfusion injury.

Topics: Animals; Butadienes; Cell Survival; Coculture Techniques; Endothelial Cells; Endothelium, Vascular; Enzyme Inhibitors; Female; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Isoflurane; L-Lactate Dehydrogenase; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase Type III; Nitriles; Oxidation-Reduction; Phosphorylation; Protein Transport; Rats; Reperfusion Injury; Signal Transduction; Up-Regulation

We previously demonstrated that hypoxia increased the hypoxia-inducible factor (HIF-1)-dependent MGr1-Ag/37LRP expression, which enhanced adhesion of gastric cancer cells to laminin, inhibited drug-induced apoptosis and caused cell adhesion-mediated drug resistance (CAM-DR). Here, we investigated the role of extracellular-regulated kinase (ERK) 1/2 in the signaling mechanisms underlying these events. We found that hypoxia activated ERK activity in vitro and in vivo. Overexpression of mitogen-activated protein kinase (MAPK) kinase (MEK), which preferentially activated ERK, mimics, in a nonadditive way, hypoxia-induced activity of MGr1-Ag/37LRP promoter and expression of MGr1-Ag/37LRP. Furthermore, U0126, the MEK inhibitor, inhibited hypoxia- and MEK-induced MGr1-Ag/37LRP promoter activity in a dose-dependent manner. MEK inhibition also reversed hypoxia- and MEK-induced HIF-1 protein and its activity in a dose-dependent manner. We also investigated reactive oxygen species signaling this response. Exogenous addition of H(2)O(2) was sufficient to activate ERK in a dose-dependent profile. Reactive oxygen species scavengers of H(2)O(2) significantly inhibited hypoxia-induced ERK or HIF-1 activation and sequential expression of MGr1-Ag/37LRP. We also investigated the signaling in hypoxia-induced cell adhesion and apoptosis induced by vincristine. Hypoxia significantly enhanced adhesion of SGC7901 cells to laminin in a time-dependent manner, which might be inhibited by the MEK inhibitor U0126 and MGr1-Ag/37LRP siRNA. Consistent with results of adhesion assay, hypoxia-resistant apoptosis might be reversed by U0126 in a dose-dependent manner. Our results suggest that hypoxia-elicited MGr1-Ag/37LRP expression activated by HIF-1 depends on ERK activation. These events are dependent of reactive oxygen intermediates.

Topics: Animals; Antigens, Neoplasm; Apoptosis; Blotting, Western; Butadienes; Cell Adhesion; Cell Movement; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Hydrogen Peroxide; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Oxidants; Phosphorylation; Promoter Regions, Genetic; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Stomach Neoplasms; Tumor Cells, Cultured

Transplantation of embryonic stem (ES) cell-derived neural progenitor cells (ES-NPCs) is one promising technology for the treatment of spinal cord injury. Promoting ES-NPC survival at the lesion site is critical for the successful treatment. We tested the role of insulin in promoting mouse ES-NPC survival. Cultured ES-NPCs survived when maintained in normoxia but underwent apoptosis when exposed to hypoxia. Insulin rescued ES-NPCs from hypoxia-induced cell death. This effect could be blocked by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor LY294002. In contrast, mitogen-activated protein kinase (MAP)/extracellular-signal-regulated kinase (ERK) pathway inhibitor U0126 potentiated insulin-mediated survival. Immunoblots revealed that insulin upregulated activation of Akt and inhibited ERK activation through the PI3K pathway. In addition, we showed that insulin reduced the activation of caspase-3, the key executor of apoptosis. In summary, our data suggest that insulin prevent apoptosis in ES-NPCs by activating Akt and inhibiting ERK through the PI3K pathway.

Topics: Animals; Apoptosis; Butadienes; Cell Differentiation; Cells, Cultured; Chromones; Drug Interactions; Embryo, Mammalian; Embryonic Stem Cells; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Hypoglycemic Agents; Hypoxia; Insulin; Mice; Morpholines; Nerve Tissue Proteins; Neurons; Nitriles; Oncogene Protein v-akt; Protein Serine-Threonine Kinases; Signal Transduction

Macrophages exposed to hyperoxia in the lung continue to survive for prolonged periods. We previously reported (Nyunoya, T., Powers, L. S., Yarovinsky, T. O., Butler, N. S., Monick, M. M., and Hunninghake, G. W. (2003) J. Biol. Chem. 278, 36099-36106) that hyperoxia induces cell cycle arrest and sustained extracellular signal-related kinase (ERK) activity in macrophages. In this study, we determined the mechanisms of hyperoxia-induced ERK activation and how ERK activity plays a pro-survival role in hyperoxia-exposed cells. Inhibition of ERK activity decreased survival of hyperoxia-exposed macrophages. This was due, at least in part, to down-regulation of the pro-apoptotic Bcl-2 family member, BimEL. In determining the mechanism of ERK activation by hyperoxia, we found that ERK activation was not associated with hyperoxia-induced activation of the upstream ERK kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2. When we examined the ability of whole cell lysates from hyperoxia-exposed cells to dephosphorylate purified phosphorylated ERK, we found decreased ERK-directed phosphatase activity. Two particular ERK-directed phosphatases (protein phosphatase 2A and MAPK phosphatase-3) demonstrated decreased activity in hyperoxia-exposed cells. Moreover, whole cell lysates from normoxia-exposed cells depleted of PP2A or MAPK phosphatase-3 were also less able to dephosphorylate ERK. These data demonstrate that, in hyperoxia-exposed macrophages, sustained activation of ERK due to phosphatase down-regulation permits macrophage survival via effects on the balance between pro- and anti-apoptotic Bcl-2 family proteins.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Blotting, Western; Butadienes; Cell Survival; Cells, Cultured; Down-Regulation; Dual Specificity Phosphatase 6; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hypoxia; Macrophages; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Membrane Proteins; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Nitriles; Phosphoprotein Phosphatases; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Phosphatase 2; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Serine; Time Factors

We have previously demonstrated that Bcl-2 overexpression in human breast carcinoma and melanoma cells synergizes with hypoxia to increase angiogenesis through up-regulation of vascular endothelial growth factor. In this work we demonstrated, for the first time, that Bcl-2 overexpression in cancer cells exposed to hypoxia modulates urokinase plasminogen activator receptor (uPAR) expression through Sp1 transcription factor and that the extracellular signal-regulated kinase (ERK) pathway plays a role in Sp1 transcriptional activity. In particular, an increase in uPAR protein and mRNA expression was found in melanoma bcl-2 transfectants grown under hypoxia when compared with control cells, and a decrease of uPAR protein expression was induced by treatment of cells with specific bcl-2 antisense oligonucleotides. Up-regulation of uPAR expression was accompanied by increased Sp1 protein expression, stability, serine phosphorylation, and DNA binding activity. Treatment of cells with mitramycin A, an inhibitor of Sp1 activity, confirmed the role of Sp1 transcriptional activity in uPAR induction by Bcl-2. The contribution of the ERK pathway in Sp1-increased transcriptional activity was demonstrated by the use of chemical inhibition. In fact, ERK kinase activation was induced in Bcl-2-overexpressing cells exposed to hypoxia, and the ERK kinase inhibitor UO126 was able to down-regulate Sp1 phosphorylation and DNA binding activity. Using a human breast carcinoma line, we obtained data supporting our findings with melanoma cells and identified a link between the induction of Sp1 and uPAR expression as a common bcl-2-controlled phenomenon in human tumors. In conclusion, our results strongly indicate that up-regulation of uPAR expression by Bcl-2 in hypoxia is modulated by Sp1 DNA binding activity through the ERK signaling pathway.

Topics: Blotting, Northern; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Nucleus; DNA; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Hypoxia; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Oligonucleotides, Antisense; Phosphorylation; Plicamycin; Precipitin Tests; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-2; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Serine; Signal Transduction; Sp1 Transcription Factor; Time Factors; Transcription, Genetic; Transfection; Up-Regulation

Hypoxia, frequently found in the center of solid tumor, is associated with resistance to chemotherapy by activation of signaling pathways that regulate cell pro-liferation, angiogenesis, and apoptosis. We determined whether hypoxia can increase the resistance of human pancreatic carcinoma cells to gemcitabine-induced apoptosis by activation of phosphatidylinositol 3'-kinase (PI3K)/Akt, MEK/mitogen-activated protein kinase (extracellular signal-regulated kinase) [MAPK(Erk) kinase (MEK)], and nuclear factor kappa B (NF-kappa B) signaling pathways.. We evaluated the phosphorylation of Akt and MAPK(Erk), DNA binding activity of NF-kappa B, and apoptosis induced by gemcitabine in L3.6pl human pancreatic cancer cells under normoxic and hypoxic conditions. We then examined the effects of the PI3K inhibitor LY294002, MEK inhibitor U0126, and the epidermal growth factor receptor tyrosine kinase inhibitor PKI 166 on these signaling pathways and induction of apoptosis.. Hypoxic conditions increased phosphorylation of Akt and MAPK(Erk) and NF-kappa B DNA binding activity in L3.6pl cells. The activation of Akt and NF-kappa B was prevented by LY294002, whereas the activity of MAPK(Erk), but not NF-kappa B, was inhibited by U0126. The increased activation of Akt, NF-kappa B, and MAPK(Erk) was inhibited by PKI 166. Under hypoxic conditions, L3.6pl cells were resistant to apoptosis induced by gemcitabine. The addition of LY294002 or PKI 166 abrogated cell resistance to gemcitabine, whereas U0126 only partially decreased this resistance.. These data demonstrate that hypoxia can induce resistance of pancreatic cancer cells to gemcitabine mainly through the PI3K/Akt/NF-kappa B pathways and partially through the MAPK(Erk) signaling pathway. Because PKI 166 prevented the activation of PI3K/Akt/NF-kappa B and MAPK(Erk) pathways, the combination of this tyrosine kinase inhibitor with gemcitabine should be an effective therapy for pancreatic cancer.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Blotting, Western; Butadienes; Cell Division; Cell Line, Tumor; Chromones; Deoxycytidine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gemcitabine; Humans; Hypoxia; Mitogen-Activated Protein Kinases; Morpholines; Neovascularization, Pathologic; NF-kappa B; Nitriles; Oxygen; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein-Tyrosine Kinases; Pyrimidines; Pyrroles; Signal Transduction; Sp1 Transcription Factor; Time Factors; Tyrosine

Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases.

Topics: Adenoviridae; Angiopoietin-2; Animals; Blotting, Northern; Blotting, Western; Butadienes; Carbazoles; Cattle; Cell Division; Disease Models, Animal; DNA; Enzyme Inhibitors; Flavonoids; Genes, Dominant; Humans; Hypoxia; Indoles; Maleimides; Mice; Models, Biological; Neovascularization, Pathologic; Neuropilin-1; Nitriles; Phosphorylation; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Reperfusion Injury; Retina; RNA, Messenger; Staurosporine; Time Factors; Transcription Factors; Up-Regulation; Vascular Endothelial Growth Factor A